Biological Activities of Extracts from Ageratum fastigiatum: Phytochemical Study and In Silico Target Fishing Approach.

In the present study, the ethanolic extract from aerial parts of Ageratum fastigiatum was evaluated in vitro against epimastigote forms of Trypanosoma cruzi (Y strain), promastigote forms of Leishmania amazonensis (PH8 strain), and L. chagasi (BH400 strain). The extract was also evaluated against Staphylococcus aureus (ATCC 25 923), Escherichia coli (ATCC 11 775), Pseudomonas aeruginosa (ATCC 10 145), and Candida albicans (ATCC 36 802). The phytochemical screening was performed by thin-layer chromatography and high-performance liquid chromatography. The extract was fractionated using flash preparative chromatography. The ethanolic extract showed activity against T. cruzi, L. chagasi, and L. amazonensis and antimicrobial activity against S. aureus, E. coli, P. aeruginosa, and C. albicans. The phytochemical screening revealed coumarins, terpenes/sterols, and flavonoids in the ethanolic extract. In addition, the coumarin identified as ayapin was isolated from this extract. We also performed in silico prediction of potential biological activities and targets for compounds previously found in A. fastigiatum. Several predictions were confirmed both retrospectively and prospectively by experimental results described here or elsewhere. Some activities described in the in silico target fishing approach were validated by the ethnopharmacological use and known biological properties. Some new activities and/or targets were predicted and could guide future studies. These results suggest that A. fastigiatum can be an interesting source of substances with antiparasitic and antimicrobial activities.

Efficacy and safety in the treatment of hyperprolactinemia: A systematic review and network meta-analysis.

WHAT IS KNOWN AND OBJECTIVE: Hyperprolactinemia is a neuroendocrine disease that is responsible for a quarter of cases of secondary amenorrhea, which can lead to infertility in women. Dopaminergic agonists (bromocriptine, cabergoline, quinagolide) can be used in the treatment. However, there is a lack of secondary studies that compare their efficacy and safety, especially through a network meta-analysis. Thus, to contribute to the decision-making, a systematic review and network meta-analyses (NMA) were performed to evaluate the efficacy and safety of dopaminergic agonists in the treatment of hyperprolactinemia. METHODS: Randomized clinical trials (RCT) were retrieved through PubMed, Web of Science and Scopus databases. The efficacy and safety of the drugs were compared, considering the following outcomes: prolactin (PRL) levels, number of patients with galactorrhoea, menstrual irregularities and adverse drug reactions. NMA was built for each outcome. Results were reported as odds ratios (OR) with 95% credibility intervals. Ranking probabilities were calculated by surface under the cumulative ranking analysis (SUCRA) and Stochastic multicriteria acceptability analysis (SMAA). RESULTS AND DISCUSSION: Seventeen RCTs were included in the systematic review and fifteen in the meta-analyses. The drugs had similar efficacy, considering the PRL levels. The SUCRA analysis showed that quinagolide (0.075 and 0.05 mg/day) was superior for reducing irregular menstruation, whereas bromocriptine was the best (97%) for galactorrhoea. Cabergoline proved to be the safest drug, except for abdominal pain at a dose of 1 mg/week. The SMAA demonstrated similar results to SUCRA. WHAT IS NEW AND CONCLUSION: This is the first network meta-analysis that evaluated the efficacy and safety of dopaminergic agonists in the treatment of hyperprolactinemia. The results of this review revealed that these drugs have similar efficacy, but cabergoline has a better safety profile.

Myofibroblastic lesions in the oral cavity: Immunohistochemical and ultrastructural analysis.

OBJECTIVE: To immunohistochemically characterize a group of oral myofibroblastic lesions (MLs) and to evaluate the ultrastructural features of myofibroblasts. MATERIAL AND METHODS: Using a tissue microarray technique (TMA), cases of myofibroma (MF), of nodular fasciitis (NF), of desmoplastic fibroma (DF), and of myofibroblastic sarcoma (MS) from the Universidad Autónoma Metropolitana Xochimilco, and a Private Oral Pathology Service in Mexico City were stained with antibodies against alpha-smooth muscle actin (α-SMA), H-caldesmon, vimentin, desmin, ß-catenin, CD34, anaplastic lymphoma protein kinase (ALK-1), and Ki-67. RESULTS: Nineteen of the 22 MF cases, 2/5 of the NF cases, 1/10 of the DF cases, and 1/2 of the MS cases were positive for α-SMA. 1/2 of the MS cases were positive for desmin; 6/10 of the DF cases were positive for ß-catenin, and 2 of the MF cases were positive for ALK-1. All of the MLs were positive for vimentin and negative for H-caldesmon and CD-34. The Ki-67 labeling index in all of the 8/22 MF, 3/5 NF, and 2/2 MS cases was ≥10%. For all of the MLs evaluated, ultrastructural analysis revealed spindle-shaped cells containing endoplasmic reticulum and peripheral actin filament bundles. CONCLUSION: In certain myofibroblastic lesions, the use of auxiliary techniques (such as immunohistochemistry) can be critical for differential diagnosis.

The role of mitochondrial DNA damage at skeletal muscle oxidative stress on the development of type 2 diabetes.

Reduced cellular response to insulin in skeletal muscle is one of the major components of the development of type 2 diabetes (T2D). Mitochondrial dysfunction involves in the accumulation of toxic reactive oxygen species (ROS) that leads to insulin resistance. The aim of this study was to verify the involvement of mitochondrial DNA damage at ROS generation in skeletal muscle during development of T2D. Wistar rats were fed a diet containing 60% fat over 8 weeks and at day 14 a single injection of STZ (25 mg/kg) was administered (T2D-induced). Control rats received standard food and an injection of citrate buffer. Blood and soleus muscle were collected. Abdominal fat was quantified as well as glucose, triglyceride, LDL, HDL, and total cholesterol in plasma and mtDNA copy number, cytochrome b (cytb) mRNA, 8-hydroxyguanosine, and 8-isoprostane (a marker of ROS) in soleus muscle. T2D-induced animal presented similar characteristics to humans that develop T2D such as changes in blood glucose, abdominal fat, LDL, HDL and cholesterol total. In soleus muscle 8-isoprostane, mtDNA copy number and 8-hydroxyguanosine were increased, while cytb mRNA was decreased in T2D. Our results suggest that in the development of T2D, when risks factors of T2D are present, intracellular oxidative stress increases in skeletal muscle and is associated with a decrease in cytb transcription. To overcome this process mtDNA increased but due to the proximity of ROS generation, mtDNA remains damaged by oxidation leading to an increase in ROS in a vicious cycle accounting to the development of insulin resistance and further T2D.

An update of 77 cases diagnosed as oral hemangiomas based on GLUT-1 positivity.

OBJECTIVES: To evaluate cases diagnosed as "oral hemangiomas" based on the immunohistochemical expression of human glucose transporter protein (GLUT-1) and on histopathological features, and to investigate whether the classification proposed by the ISSVA was used correctly to classify these lesions. MATERIAL AND METHODS: All cases stored in the archives of an Oral Pathology Service and diagnosed as "oral hemangiomas" were reviewed. Seventy-seven cases were analyzed regarding the expression of GLUT-1. GLUT-1(+) specimens were classified as true infantile hemangioma (IH) and GLUT-1(-) specimens were reclassified based on their histopathological features. The nomenclature of these lesions was evaluated and some cases were reclassified. RESULTS: Only 26 (33.8%) of the specimens were indeed IHs. Among the GLUT-1(-) specimens, 20 (26.0%) were reclassified as pyogenic granulomas (PGs) and 31 (40.2%) as vascular malformations. Considering the previously applied nomenclature, only 47.5% of the cases initially diagnosed as "hemangiomas" were IHs. In the group of "capillary hemangiomas", most cases (56.2%) were PGs. Among the three "cellular hemangiomas", two were PGs and one was IH. Most (88.8%) "cavernous hemangiomas" were vascular malformations. CONCLUSION: Careful and parameterized review of cases of vascular anomalies is necessary using auxiliary tools such as GLUT-1, since the exclusive use of histopathological findings might be insufficient to differentiate some anomalies. CLINICAL RELEVANCE: Accurate clinical examination and the use of biomarkers such as GLUT-1 are essential for the diagnosis.

Effect of potentially chromogenic beverages on shear bond strength of acrylic denture teeth to heat-polymerized denture base resins.

BACKGROUND: Detachment of denture acrylic resin artificial teeth from denture base resin is one of the most common problems presented by denture wearers. PURPOSE: This study investigated the shear bond strength (SBS) and fracture type of bonding interface of two commercial acrylic teeth (Vipi Dent Plus e Biolux) to two denture base resins (Vipi Cril e Lucitone 550) after immersion in potentially chromogenic beverages (coffee, cola soft drink, and red wine) or control solution (distilled water). MATERIALS AND METHODS: Maxillary central incisor acrylic teeth were placed at 45° to denture base resin and submitted to short polymerization cycle according to manufacturers. Specimens were divided according to the combination tooth/resin/solution (n = 8) and submitted to bond strength tests in a universal testing machine MTS-810 (0.5 mm/min). Subsequently, fracture area was analyzed by stereomicroscope at a magnification of ×10 and categorized into adhesive, cohesive, or mixed failure. RESULTS: The bond strength of teeth/denture base resins interface was not significantly affected by tested solutions (P > 0.087), except for Biolux teeth immersed in coffee (P < 0.01). In all conditions, the Vipi Dent Plus teeth showed higher bond strength to Lucitone and Vipi Cril resins when compared to Biolux teeth (P < 0.003). All specimens' failure modes were cohesive. CONCLUSIONS: The SBS of acrylic teeth to denture base resins was not generally influenced by immersion in the tested staining beverages.

Distribution of Hypoxia-Inducible Factor-1α and Glucose Transporter-1 in Human Tongue Cancers.

PURPOSE: Oral squamous cell carcinomas have the potential for rapid and unlimited growth. Therefore, hypoxic tissue areas are common in these malignant tumors and contribute to cancer progression, therapy resistance, and poor outcomes. The aim of the present study was to analyze the gene product distribution of hypoxia-inducible factor-1α (HIF-1α) and glucose transporter-1 (GLUT-1) in cases of tongue squamous cell carcinoma (TSCC) and to identify a preliminary correlation between these proteins and clinical staging and Brynes's histologic grading system (HGS). MATERIALS AND METHODS: The sample included 57 cases of TSCC. Histologic sections of 3 µm were submitted to the immunoperoxidase method and semiquantitative analysis. The association between HIF-1α and GLUT-1 expression in TSCC and the clinical stage and the HGS of Bryne (1998) was evaluated using the χ(2) test, with the significance level set at 0.05 (α = 0.05). RESULTS: HIF-1α was mainly expressed in the nucleus/cytoplasm of neoplastic cells, most specimens exhibited diffuse staining in neoplastic cells (84.2%), and focal staining was only observed in perinecrotic areas (15.8%). GLUT-1 was expressed in the cytoplasm and membrane of malignant cells, and diffuse staining was observed in all cases. The intensity of HIF-1α expression correlated significantly with clinical stage (P = .011) and HGS (P = .002). A significant association was observed between the distribution of HIF-1α expression and metastasis (P = .040). Immunoexpression of GLUT-1 correlated significantly with clinical stage (P = .002) and HGS (P = .000). GLUT-1 expression in the peripheral island was predominant in most low-grade tumors (78.6%); in the high-grade cases, the expression prevailed in the location center/periphery (55.8%). Comparison of the location of the tumor island in the different histologic grades showed a statistically significant difference (P = .025). CONCLUSION: The expression of HIF and GLUT proteins within TSCC appears to be associated with disease stage, grade, and the presence of metastases. Additional studies are needed to evaluate the diagnostic and prognostic uses of these proteins in the treatment of TSCC.

Antifungal activity of 4% chlorhexidine and 2% sodium hypochlorite against Candida albicans biofilms.

The purpose of this study was to determine the antifungal efficacy of 4% chlorhexidine (CHX) and 2% sodium hypochlorite (NaOCl) on 24-hour Candida albicans biofilms. Candida albicans biofilms were developed on acrylic resin specimens, which were randomly assigned to 1 of 5 groups (n = 3 per group) exposed to 1 mL of 4% CHX for 2, 4, 6, 8, or 10 minutes. Biofilms in contact with 1 mL of distilled water or 2% NaOCl for 10 minutes were used as positive and negative controls (n = 3 per group), respectively. Specimens were analyzed with confocal laser scanning microscopy and a cell viability assay technique. The biovolume of the live subpopulation of the biofilm was calculated with biofilm image analysis software. Statistically significant differences (P < 0.05) in the biovolume of surviving cells were found among the positive control group and the 4-, 6-, 8-, and 10-minute experimental periods. The biovolumes found after 6-10 minutes of exposure to chlorhexidine were significantly different (P < 0.05) from the biovolume after 2 minutes of exposure. The most effective decrease of the biovolume was found after the use of the negative control (sodium hypochlorite) solution. Exposure to 10 minutes of 2% NaOCl removed fungal cells more effectively than all the experimental groups (P < 0.05). The 4% CHX solution showed an antifungal activity against C albicans biofilms but failed to decrease the biovolume to the levels of 2% NaOCl, which eliminated viable cells more effectively and appeared to be more effective in disrupting the attached biofilms.

Study of the etiopathogenesis and differential diagnosis of oral vascular lesions by immunoexpression of GLUT-1 and HIF-1α.

BACKGROUND: To investigate whether the immunohistochemical expression of GLUT-1 and HIF-1α is related to the diagnosis and pathogenesis of oral vascular lesions. STUDY METHODS: Thirty cases each of pyogenic granuloma (PG) and hemangioma were studied. Antibodies against GLUT-1 and HIF-1α were detected by immunoperoxidase staining in 3-µm histological sections, and the results were analyzed qualitatively and quantitatively, respectively. Positive and negative cells were counted, and the mean number of positive cells was calculated for each case. RESULTS: The initial diagnosis of hemangioma was maintained in only 7 (23%) of the 30 cases studied, which were positive for GLUT-1. The remaining 23 cases were reclassified as vascular malformation (VM) (n = 13) and PG (n = 10) due to the absence of staining. The endothelium of blood vessels was negative for GLUT-1 in all cases initially diagnosed as PG (n = 30). The percentage of HIF-1α-positive cells was higher in cases of PG, followed by hemangiomas and VMs (P = 0.005). CONCLUSIONS: Histological features are not sufficient to establish the correct diagnosis of oral hemangiomas, and an accurate anamnesis is essential in these cases. In addition, these findings demonstrate that vascular lesions express mediators of angiogenesis, HIF-1α, and suggest that his process may play a role in the pathogenesis of vascular.

Relationship between the epithelial expression of hMLH1, MDM2, and p63 and lower lip carcinogenesis.

OBJECTIVES: To evaluate the relationship between the epithelial expression of hMLH1, MDM2, and p63 in lower lip carcinogenesis, comparing the immunostaining of these proteins in cases of actinic cheilitis (AC) and lower lip squamous cell carcinoma (SCC). STUDY DESIGN: Forty cases of AC and 40 cases of SCC were studied, both lesions were of lower lip. Histological sections of 3 µm were submitted to immunoperoxidase method, and 1000 cells were counted for immunohistochemical analysis of lesions. The results were analyzed quantitatively, and expression was compared by the Mann-Whitney, Student t-test, or one-way ANOVA, adopting a level of significance of 5%. RESULTS: A higher percentage of epithelial cells expressing hMLH1 was observed in cases of AC without dysplasia or mild dysplasia (721.23 ± 88.116), whereas fewer positive cells were observed in lower lip SSCs (255.03 ± 199.47) when compared to the AC group (P < 0.001). Immunoexpression of MDM2 was higher in SCCs of the lower lip compared with AC (P = 0.019). For p63 protein, the expression was higher in AC than in SCC (P = 0.045). CONCLUSION: The present results showed changes in the immunoexpression of hMLH1, MDM2, and p63 in epithelial cells from premalignant and malignant lip disease, supporting the hypothesis that these alterations are related to the process of lower lip carcinogenesis.

Long­term effects of vitexin against development of pentylenetetrazole­induced kindling in rats.

Evidence is provided that the glycosylated flavonoid vitexin (apigenin­8­C­beta­D­glucopyranoside) attenuates pentylenetetrazole (PTZ)­induced acute tonic­clonic seizures in rats. However, the effects of chronic and systemic vitexin in PTZ­kindled rats remain unknown. The aim of this work was to investigate the effect of long­term treatment with vitexin in the PTZ­kindling model of epilepsy. Male Wistar rats received intraperitoneal injections of PTZ at a subconvulsive dose of 35 mg/kg every other day for 29 days. Either saline containing dimethyl sulfoxide - DMSO 1%  (vehicle), diazepam (2 mg/kg; positive control) or vitexin (2.5 mg/kg) was administered intraperitoneally 30 min before each PTZ injection. The behavioral reactions were recorded by 30 min immediately after each PTZ injection. Furthermore, on the 31st day, that is, 48 h after the latter dose of PTZ, the animals were euthanized and renal and hepatic biochemical markers were evaluated in blood serum. Chronic treatment with either diazepam or vitexin attenuated the seizures provoked by PTZ injections. Neither diazepam nor vitexin caused changes in renal levels of creatinine and urea and in hepatic levels of aspartate aminotransferase and alanine aminotransferase. Our findings suggest that chronic administration of vitexin attenuates the progression of PTZ­induced kindling without causing side effects on kidneys and liver.

Zinc, antioxidant enzymes in preeclampsia, and association with newborn outcome.

BACKGROUND: The etiology of preeclampsia (PE) may be associated with the increased of production of reactive species and decreased antioxidant activity of enzymes. Inadequate intake of Zn can affect gestational health due to its biological functions, such as its role in the antioxidant defense system. The study aimed to assess the nutritional status of Zn and antioxidant enzymes in postpartum women and its correlation with neonatal outcomes. METHODS: A cross-sectional analytical study was carried out at a referral gynecology and obstetrics hospital. A total of 119 women (PE = 58, HP = 61) participated in the study. A quantitative food-frequency questionnaire was used to assess food consumption and further analyze the dietary Zn levels. Zinc levels in plasma and erythrocytes samples were analyzed by flame atomic absorption spectrometry, catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidase (GPx) levels were determined by UV-Vis spectrophotometry. RESULTS: Plasma and dietary intake Zn results were considered adequate and without statistical difference between groups. SOD levels were significantly higher in the HP group (p = 0.011), and CAT levels were higher in the PE group (p = 0.050). There was a positive correlation between SOD activity in women with PE and the weight of their newborns (r = 0.336, p=0.021). CONCLUSION: The results showed adequate Zn levels (consumption and serum levels) in the groups studied, although with a reduction of plasma Zn in the PE group compared to the PH group. Zinc in plasma fractions and erythrocytes are important markers for oxidative stress, in particular, plasma Zn seems to be related to the rapid response to preeclampsia. The activity of antioxidant enzymes was elevated in the groups studied. Better SOD activity improves birth weight in children of pregnant women with preeclampsia.

Distinct anti-NP, anti-RBD and anti-Spike antibody profiles discriminate death from survival in COVID-19.

Introduction: Infection by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) induces rapid production of IgM, IgA, and IgG antibodies directed to multiple viral antigens that may have impact diverse clinical outcomes. Methods: We evaluated IgM, IgA, and IgG antibodies directed to the nucleocapsid (NP), IgA and IgG to the Spike protein and to the receptor-binding domain (RBD), and the presence of neutralizing antibodies (nAb), in a cohort of unvaccinated SARS-CoV-2 infected individuals, in the first 30 days of post-symptom onset (PSO) (T1). Results: This study included 193 coronavirus disease 2019 (COVID-19) participants classified as mild, moderate, severe, critical, and fatal and 27 uninfected controls. In T1, we identified differential antibody profiles associated with distinct clinical presentation. The mild group presented lower levels of anti-NP IgG, and IgA (vs moderate and severe), anti-NP IgM (vs severe, critical and fatal), anti-Spike IgA (vs severe and fatal), and anti-RBD IgG (vs severe). The moderate group presented higher levels of anti-RBD IgA, comparing with severe group. The severe group presented higher levels of anti-NP IgA (vs mild and fatal) and anti-RBD IgG (vs mild and moderate). The fatal group presented higher levels of anti-NP IgM and anti-Spike IgA (vs mild), but lower levels of anti-NP IgA (vs severe). The levels of nAb was lower just in mild group compared to severe, critical, and fatal groups, moreover, no difference was observed among the more severe groups. In addition, we studied 82 convalescent individuals, between 31 days to 6 months (T2) or more than 6 months (T3), PSO, those: 12 mild, 26 moderate, and 46 severe plus critical. The longitudinal analyzes, for the severe plus critical group showed lower levels of anti-NP IgG, IgA and IgM, anti-Spike IgA in relation T3. The follow-up in the fatal group, reveals that the levels of anti-spike IgG increased, while anti-NP IgM levels was decreased along the time in severe/critical and fatal as well as anti-NP IgG and IgA in several/critical groups. Discussion: In summary, the anti-NP IgA and IgG lower levels and the higher levels of anti-RBD and anti-Spike IgA in fatal compared to survival group of individuals admitted to the intensive care unit (ICU). Collectively, our data discriminate death from survival, suggesting that anti-RBD IgA and anti-Spike IgA may play some deleterious effect, in contrast with the potentially protective effect of anti-NP IgA and IgG in the survival group.

[Nursing care management in hospital settings: the building of a construct]. / Gerência do cuidado de enfermagem em cenários hospitalares: a construção de um conceito.

The objective of this study was to build and present a theoretical definition of the concept of nursing care management in hospital settings, based on specific literature. We chose to use Concept Analysis strategies for building concepts, the rules of Archeological Analysis for forming concepts, and Lexical Analysis as the theoretical-methodological framework. The operationalization of the strategies and rules for forming the concept permitted the construction of the concept of nursing care in hospital settings. The constructed concept presented, by its nature, the capacity to form a dialectic integration of the aspects relative to the knowing-doing of care and management. The theoretical definition of the concept of Nursing Care Management in Hospital Settings assigned meaning to the term, in the initial context of the construction of a theory of Nursing Care Management in Health Services.

Iron toxicity increases oxidative stress and impairs mineral accumulation and leaf gas exchange in soybean plants during hypoxia.

Iron toxicity is a major challenge faced by plants in hypoxic soils; however, the consequences of such combined stress for soybean (Glycine max) remain to be determined. Here we assessed the physiological responses of soybean plants exposed to hypoxia and a high concentration of iron. Soil-grown plants cultivated in a greenhouse until the vegetative stage were transferred to a hydroponic system containing nutrient solution and subjected to two oxygen conditions (normoxia (6.2 mg L-1) and hypoxia (0.33 mg L-1)) and two iron concentrations (Fe-EDTA) (0.09 and 1.8 mM) for 72 h. During hypoxia, high concentrations of iron in the nutrient solution resulted in increased iron accumulation in roots and leaves. Under this condition, the concentrations of zinc, nitrogen, potassium, and calcium decreased in the roots, while the concentration of nitrogen and magnesium decreased in the leaves. Additionally, during hypoxia, the higher concentration of iron led to an increase in the activity of the antioxidant enzymes in roots and leaves, while decreased the levels of the photosynthetic pigments, leaf gas exchange, and plant growth. In conclusion, high iron concentration in the root medium results in a considerably more severe damage condition to soybean plants under hypoxia compared to plants grown under low iron availability.

Waterlogging priming alleviates the oxidative damage, carbohydrate consumption, and yield loss in soybean (Glycine max) plants exposed to waterlogging.

In this study, we tested whether waterlogging priming at the vegetative stage would mitigate a subsequent waterlogging event at the reproductive stage in soybean [Glycine max (L.) Merr.]. Plants (V3 stage) were subjected to priming for 7days and then exposed to waterlogging stress for 5days (R2 stage) with non-primed plants. Roots and leaves were sampled on the fifth day of waterlogging and the second and fifth days of reoxygenation. Overall, priming decreased the H2 O2 concentration and lipid peroxidation in roots and leaves during waterlogging and reoxygenation. Priming also decreased the activity of antioxidative enzymes in roots and leaves and increased the foliar concentration of phenols and photosynthetic pigments. Additionally, priming decreased fermentation and alanine aminotransferase activity during waterlogging and reoxygenation. Finally, priming increased the concentration of amino acids, sucrose, and total soluble sugars in roots and leaves during waterlogging and reoxygenation. Thus, primed plants were higher and more productive than non-primed plants. Our study shows that priming alleviates oxidative stress, fermentation, and carbohydrate consumption in parallel to increase the yield of soybean plants exposed to waterlogging and reoxygenation.

Senescence State in Mesenchymal Stem Cells at Low Passages: Implications in Clinical Use.

Mesenchymal stem cells (MSCs) are multipotent cells found in various tissues and are easily cultivated. For use in clinical protocols, MSCs must be expanded to obtain an adequate number of cells, but a senescence state may be instituted after some passages, reducing their replicative potential. In this study, we report a case where MSC derived from an elderly donor acquired a senescence state after three passages. The bone marrow was aspirated from a female patient submitted to a cell therapy for the incontinency urinary protocol; MSCs were cultivated with DMEM low glucose, supplemented with 10% autologous serum (AS) plus 1% L-glutamine and 1% antibiotic/antimycotic. Senescence analysis was performed by ß-galactosidase staining after 24 and 48 h. Controls were established using BM-MSC from healthy donors and used for senescence and gene expression assays. Gene expression was performed using RT-PCR for pluripotency genes, such as SOX2, POU5F1, NANOG, and KLF4. MSC telomere length was measured by the Southern blotting technique, and MSCs were also analyzed for their capacity to differentiate into adipocytes, chondrocytes, and osteocytes. The patient's MSC expansion using AS displayed an early senescence state. In order to understand the role of AS in senescence, MSCs were then submitted to two different culture conditions: 1) with AS or 2) with FBS supplementation. Senescence state was assessed after 24 h, and no statistical differences were observed between the two conditions. However, patients' cells cultured with AS displayed a higher number of senescence cells than FBS medium after 48 h (p = 0.0018). Gene expression was performed in both conditions; increased expression of KLF4 was observed in the patient's cells in comparison to healthy controls (p = 0.0016); reduced gene expression was observed for NANOG (p = 0.0016) and SOX2 (p = 0.0014) genes. Telomere length of the patient's cells was shorter than that of a healthy donor and that of a patient of similar age. Osteocyte differentiation seemed to be more diffuse than that of the healthy donor and that of the patient of similar age. MSCs could enter a senescence state during expansion in early passages and can impact MSC quality for clinical applications, reducing their efficacy when administered.

An inter-laboratory study of DNA-based identity, parentage and species testing in animal forensic genetics.

The probative value of animal forensic genetic evidence relies on laboratory accuracy and reliability. Inter-laboratory comparisons allow laboratories to evaluate their performance on specific tests and analyses and to continue to monitor their output. The International Society for Animal Genetics (ISAG) administered animal forensic comparison tests (AFCTs) in 2016 and 2018 to assess the limitations and capabilities of laboratories offering forensic identification, parentage and species determination services. The AFCTs revealed that analyses of low DNA template concentrations (≤300 pg/µL) constitute a significant challenge that has prevented many laboratories from reporting correct identification and parentage results. Moreover, a lack of familiarity with species testing protocols, interpretation guidelines and representative databases prevented over a quarter of the participating laboratories from submitting correct species determination results. Several laboratories showed improvement in their genotyping accuracy over time. However, the use of forensically validated standards, such as a standard forensic short tandem repeat (STR) kit, preferably with an allelic ladder, and stricter guidelines for STR typing, may have prevented some common issues from occurring, such as genotyping inaccuracies, missing data, elevated stutter products and loading errors. The AFCTs underscore the importance of conducting routine forensic comparison tests to allow laboratories to compare results from each other. Laboratories should keep improving their scientific and technical capabilities and continuously evaluate their personnel's proficiency in critical techniques such as low copy number (LCN) analysis and species testing. Although this is the first time that the ISAG has conducted comparison tests for forensic testing, findings from these AFCTs may serve as the foundation for continuous improvements of the overall quality of animal forensic genetic testing.

Comparison Between Nylon and Polyglactin Sutures in Pediatric Cataract Surgery: A Randomized Controlled Clinical Trial.

Purpose: To compare the performance of nylon sutures to that of polyglactin sutures in pediatric patients undergoing cataract surgery. Setting: University of Campinas (UNICAMP), Campinas, São Paulo, Brazil Design: A prospective, randomized, partially masked, single-site clinical trial. (https://clinicaltrials.gov/ct2/show/NCT03812640). Methods: A total of 80 eyes from 80 patients who underwent pediatric cataract surgery were randomized into two groups in block sizes of four. Group A consisted of 41 patients whose surgical incisions were sutured with polyglactin 10-0 material. Group B consisted of 39 patients whose surgical incisions were sutured with nylon 10-0 material. The primary outcome was frequency of suture-related complications in each group. Secondary outcomes were the frequency with which suture removal was necessary. Results: The incidence of suture-related complications within 6 months of follow up was 0 out of 41 eyes (0.00%) in the polyglactin group and 17 out of 39 eyes (43.59%) in the nylon control group (p < 0.001). In all of the eyes with suture-related complications, the sutures were promptly removed. The most frequent complications were vascularization near the suture (17.95%) and loose sutures (17.95%). No ocular or systemic study-related adverse events were observed. Conclusions: Polyglactin sutures were found to be safe and effective for pediatric patients undergoing cataract surgery. Their lower rate of complications and reduced likelihood of removal (and the subsequent need for general anesthesia) make their use preferrable to that of nylon sutures. This study represents the first controlled randomized clinical trial to compare nylon sutures to polyglactin sutures in pediatric patients undergoing cataract surgery. Clinical Trial Registration: URL: https://clinicaltrials.gov/ct2/show/, Identifier: NCT03812640.